A truncated fragment of nonmuscle myosin heavy chain II-A (NMHC II-A), lacking amino acids 1-592, has been used to examine the cellular functions of NMHC II-A. Green fluorescent protein (GFP) was fused to the N-terminal end of both full-length and truncated NMHC II-A and the fusion proteins were stably expressed in the HeLa Tet-off cell line (Clontech, Palo Alto, CA), which expresses NMHC II-A, but not NMHC II- B. The expression of truncated NMHC II-A in HeLa Tet-off cells induced cell rounding with rearrangements of actin filaments and disappearance of focal adhesions. These changes were reversed when the expression of truncated NMHC II-A was terminated by addition of doxycycline. HeLa Tet-off cells expressing the full-length NMHC II-A:GFP fusion protein did not show this phenotype with or without doxycycline. Both GFP- tagged full-length and truncated myosin was localized to actin stress fibers. However, in vitro assays showed that the baculovirus-expressed truncated myosin did not bind to actin, suggesting that the truncated myosin was brought to actin stress fibers by the endogenous myosin. Immunofluorescent studies confirmed that the truncated myosin colocalized with the endogenous myosin by using an antibody specifically recognizing only the endogenous myosin. We also demonstrated that GFP-tagged full-length myosin II-A and II-B, but not the truncated myosin, were localized to the cytokinetic ring during mitosis, indicating that the functional motor domain might be required for myosins to localize to the cytokinetic ring. - nonmuscle myosin; HeLa Tet-off; dominant negative